The present application is a divisional application of U.S. Ser. No. 09/312,266, filed May 14, 1999 now U.S. Pat. No. 6,207,879.
1. Field of the Invention
The present invention relates generally to transgenic plants. More specifically, it relates to methods and compositions for transgene expression using the maize RS81 promoter.
2. Description of the Related Art
An important aspect in the production of genetically engineered crops is obtaining sufficient levels of transgene expression in the appropriate plant tissues. In this respect, the selection of promoters for directing expression of a given transgene is crucial. Promoters which are useful for plant transgene expression include those that are inducible, viral, synthetic, constitutive as described (Poszkowski et al., 1989; Odell et al., 1985), temporally regulated, spatially regulated, and spatio-temporally regulated (Chau et al., 1989).
A number of plant promoters have been described with various expression characteristics. Examples of some constitutive promoters which have been described include the rice actin 1 (Wang et al., 1992; U.S. Pat. No. 5,641,876), CaMV 35S (Odell et al., 1985), CaMV 19S (Lawton et al., 1987), nos (Ebert et al., 1987), Adh (Walker et al., 1987), sucrose synthase (Yang and Russell, 1990).
Examples of tissue specific promoters which have been described include the lectin (Vodkin et al., 1983; Lindstrom et al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989; Dennis et al., 1984), corn light harvesting complex (Simpson, 1986; Bansal et al., 1992), corn heat shock protein (Odell et al., 1985; Rochester et al., 1986), pea small subunit RuBP carboxylase (Poulsen et al., 1986; Cashmore et al., 1983), Ti plasmid mannopine synthase (Langridge et al., 1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petunia chalcone isomerase (Van Tunen et al., 1988), bean glycine rich protein 1 (Keller et al., 1989), truncated CaMV 35s (Odell et al., 1985), potato patatin (Wenzler et al., 1989), root cell (Conkling et al., 1990), maize zein (Reina et al., 1990; Kriz et al., 1987; Wandelt and Feix, 1989; Langridge and Feix, 1983; Reina et al., 1990), globulin-1 (Belanger and Kriz et al., 1991), xcex1-tubulin, cab (Sullivan et al., 1989), PEPCase (Hudspeth and Grula, 1989), R gene complex-associated promoters (Chandler et al., 1989), and chalcone synthase promoters (Franken et al., 1991).
Inducible promoters which have been described include ABA- and turgor-inducible promoters, the promoter of the auxin-binding protein gene (Schwob et al., 1993), the UDP glucose flavonoid glycosyl-transferase gene promoter (Ralston et al., 1988); the MPI proteinase inhibitor promoter (Cordero et al., 1994), and the glyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al., 1995; Quigley et al., 1989; Martinez et al., 1989).
A class of genes which are expressed in an inducible manner are glycine-rich proteins. Expression of glycine rich proteins is induced by the plant hormone abscibic acid (ABA). Genes encoding glycine rich proteins have been described, for example, from maize (Didierjean et al., 1992; Gomez et al., 1988; Baysdorfer, Genbank Accession No. AF034945) sorghum (Cretin and Puigdomenech, 1990), and rice (Lee et al., Genbank Accession No. AF009411).
In addition to the use of a particular promoter, expression of transgenes can be influenced by other types of elements. In particular, introns have demonstrated the potential for enhancing transgene expression. For example, Callis et al. (1987) described an intron from the corn alcohol dehydrogenase gene which is capable of enhancing the expression of transgenes in transgenic plant cells. Similarly, Vasil et al. (1989) described an intron from the corn sucrose synthase gene having similar enhancing activity. The rice actin 1 intron, in particular, has found wide use in the enhancement of transgene expression in a number of different transgenic crops (McElroy et al., 1991). This 5xe2x80x2 intron was identified from the first coding exon of the rice actin 1 sequence (McElroy et al., 1990). Plant actin is encoded by a gene family present in all plant species studied to date (Meagher, 1991). In rice, there are at least eight actin-like sequences per haploid genome. Four of the rice actin coding sequences (rice actin 1, 2, 3 and 7) have been isolated and shown to differ from each other in the tissue and stage-specific abundance of their respective transcripts (Reece, 1988; McElroy et al., 1990a; Reece et al., 1990; U.S. Pat. No. 5,641,876; Genbank Accession numbers X15865, X15864, X15862, and X15863, respectively).
While the above studies have provided a number of useful tools for the generation of transgenic plants, there is still a great need in the art for novel promoter and enhancer sequences with beneficial expression characteristics. In particular, there is a need in the art for promoter-enhancer combinations which are capable of directing high-level expression of exogenous genes in transgenic crop plants. Many previously identified regulatory sequences fail to provide the levels of expression required to fully realize the benefits potentially conferred by expression of selected genes in transgenic plants. Additionally, many regulatory regions fail to demonstrate suitable or desirable expression profiles for transgene expression. There is, therefore, a great need in the art for the identification of novel sequences which can be used for the high-level expression of selected transgenes in economically important crop plants.
In one aspect, the invention provides an isolated nucleic acid comprising a maize RS81 promoter. Also provided by the invention is a maize RS81 promoter isolatable from the nucleic acid sequence of SEQ ID NO:1. The RS81 promoter may, in particular embodiments of the invention, comprise from about 50 to about 2584, from about 150 to about 2584, from about 250 to about 2584, from about 400 to about 2584, from about 750 to about 2584, from about 1000 to about 2584, from about 1500 to about 2584, or from about 2000 to about 2584, contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1. The maize RS81 promoter provided by the invention may further comprise the nucleic acid sequence of SEQ ID NO:1. In particular embodiments of the invention, the maize RS81 promoter may further comprise an enhancer, including an intron such as the rice actin 1 intron or the rice actin 2 intron. An RS81 promoter used in accordance with the invention may further include a terminator, for example, an rbcS terminator.
In another aspect, the invention provides an expression cassette comprising a maize RS81 promoter operably linked to a selected DNA. Potentially any selected DNA may be included with the expression cassette, including those encoding an insect resistance protein, a bacterial disease resistance protein, a fungal disease resistance protein, a viral disease resistance protein, a nematode disease resistance protein, a herbicide resistance protein, a protein affecting grain composition or quality, a nutrient utilization protein, a mycotoxin reduction protein, a male sterility protein, a selectable marker protein, a screenable marker protein, a negative selectable marker protein, an environment or stress resistance protein, or a protein affecting plant agronomic characteristics. The expression cassette may further include any suitable selectable marker protein. Examples of suitable selectable marker proteins include phosphinothricin acetyltransferase, glyphosate resistant EPSPS, aminoglycoside phosphotransferase, hygromycin phosphotransferase, dalapon dehalogenase, bromoxynil resistant nitrilase, anthranilate synthase and glyphosate oxidoreductase.
An expression cassette may further comprise an enhancer. In particular embodiments of the invention, the enhancer is selected from the group consisting of the rice actin 1 intron and the rice actin 2 intron. The expression cassette may still further comprise a transit peptide coding sequence, for example, a transit peptide selected from the group consisting of chlorophyll a/b binding protein transit peptide, small subunit of ribulose bisphosphate carboxylase transit peptide, EPSPS transit peptide and dihydrodipocolinic acid synthase transit peptide. In the expression cassette, the selected coding sequence may be operably linked to potentially any terminator, for example, a rice or other type of rbcS terminator.
In yet another aspect, the invention provides an expression vector comprising a maize RS81 promoter operably linked to a selected coding region. In one embodiment of the invention, the expression vector may be further defined as a plasmid vector. In another embodiment of the invention, the plasmid vector may be located within a bacterial cell.
In still yet another aspect, the invention provides a fertile transgenic plant which is stably transformed with a selected DNA comprising a maize RS81 promoter. In particular embodiments of the invention, the fertile transgenic plant comprises a maize RS81 promoter which is isolatable from the nucleic acid sequence of SEQ ID NO:1. In other embodiments of the invention, the fertile transgenic plant has a maize RS81 promoter comprising from about 50 to about 2584, from about 100 to about 2584, from about 200 to about 2584, from about 400 to about 2584, from about 750 to about 2584, from about 1000 to about 2584, from about 1500 to about 2584, or from about 2000 to about 2584, contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1. The fertile transgenic plant may also comprise the nucleic acid sequence of SEQ ID NO:1. In the fertile transgenic plant, the selected DNA may further comprise a selected coding region operably linked to said maize RS81 promoter.
The selected coding region may be potentially any protein, for example, an insect resistance protein, a bacterial disease resistance protein, a fungal disease resistance protein, a viral disease resistance protein, a nematode disease resistance protein, a herbicide resistance protein, a protein affecting grain composition or quality, a nutrient utilization protein, an environment or stress resistance protein, a mycotoxin reduction protein, a male sterility protein, a selectable marker protein, a screenable marker protein, a negative selectable marker protein, or a protein affecting plant agronomic characteristics.
Where a selected protein used in accordance with the invention is a selectable marker protein, it may be preferable to utilize a protein selected from the group consisting of phosphinothricin acetyltransferase, glyphosate resistant EPSPS, aminoglycoside phosphotransferase, hygromycin phosphotransferase, neomycin phosphotransferase, dalapon dehalogenase, bromoxynil resistant nitrilase, anthranilate synthase and glyophosate oxidoreductase. In further embodiments of the invention, the selected coding region is operably linked to a terminator, for example, a rice or other rbcS terminator. The selected DNA may also comprise an enhancer, such as a rice actin 1 intron or rice actin 2 intron. The selected DNA may comprise plasmid DNA and/or a transit peptide. In particular embodiments of the invention, the transit peptide is selected from the group consisting of chlorophyll a/b binding protein transit peptide, small subunit of ribulose bisphosphate carboxylase transit peptide, EPSPS transit peptide and dihydrodipocolinic acid synthase transit peptide.
In one embodiment of the invention, a fertile transgenic plant prepared in accordance with the invention is further defined as a monocotyledonous plant, for example, a monocot selected from the group consisting of wheat, maize, rye, rice, oat, barley, turfgrass, sorghum, millet and sugarcane. The fertile transgenic plant also may be a dicotyledonous plant, for example, a tobacco, tomato, potato, soybean, cotton, canola, alfalfa or sunflower plant. In one embodiment of the invention, the plant is a soybean plant.
In still yet another aspect, the invention provides a fertile R0 transgenic plant comprising a transgenic RS81 promoter. Also provided are seeds of the R0 transgenic plant, wherein the seeds comprise a selected DNA including the RS81 promoter. Further provided are progeny plants of any generation of the R0 transgenic plant, wherein said R0 transgenic plant comprises said selected DNA, as well as seeds of the progeny plants, wherein said seed comprises said selected DNA.
In still yet another aspect, the invention provides a crossed fertile transgenic plant prepared according to the method comprising the steps of: (i) obtaining a fertile transgenic plant comprising a selected DNA comprising a maize RS81 promoter; (ii) crossing said fertile transgenic plant with itself or with a second plant lacking said selected DNA to prepare the seed of a crossed fertile transgenic plant, wherein said seed comprises said selected DNA; and (iii) planting said seed to obtain a crossed fertile transgenic plant. The invention also provides a seed of the crossed fertile transgenic plant, wherein said seed comprises said selected DNA. In one embodiment of the invention the crossed fertile transgenic plant is a monocotyledonous plant, including a wheat, oat, barley, maize, rye, rice, turfgrass, sorghum, millet or sugarcane plant. The crossed fertile transgenic plant may also be a dicotyledonous plant, including a tobacco, tomato, potato, soybean, canola, alfalfa, sunflower or cotton plant. The selected DNA may be inherited through a male or female parent. In one embodiment of the invention, the second plant is an inbred plant, and the crossed fertile transgenic plant is a hybrid. In particular embodiments of the invention, said maize RS81 promoter is isolatable from the nucleic acid sequence of SEQ ID NO:1. In further embodiments of the invention, the maize RS81 promoter comprises from about 50 to about 2584, from about 100 to about 2584, from about 200 to about 2584, from about 400 to about 2584, from about 750 to about 2584, from about 1000 to about 2584, from about 1500 to about 2584, or from about 2000 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1. The maize RS81 promoter may also comprise the nucleic acid sequence of SEQ ID NO:1.
The crossed fertile transgenic plant may comprise a selected DNA comprising a selected coding region operably linked to the aforementioned maize RS81 promoter. In particular embodiments of the invention, the selected protein is selected from the group consisting of insect resistance protein, a bacterial disease resistance protein, a fungal disease resistance protein, a viral disease resistance protein, a nematode disease resistance protein, a herbicide resistance protein, a protein affecting grain composition or quality, a nutrient utilization protein, a mycotoxin reduction protein, a male sterility protein, a selectable marker protein, a screenable marker protein, a negative selectable marker protein, a protein affecting plant agronomic characteristics, and an environment or stress resistance protein. The selected DNA may also comprise an enhancer, such as a rice actin 1 intron or rice actin 2 intron. The selected protein may be operably linked to a terminator, for example, a rice or other rbcS terminator.
In still yet another aspect, the invention provides a method of expressing a selected protein in a transgenic plant comprising the steps of: (i) obtaining a construct comprising a selected coding region operably linked to a maize RS81 promoter; (ii) transforming a recipient plant cell with said construct; and (iii) regenerating a transgenic plant expressing said selected protein from said recipient plant cell. In one embodiment of the invention, the step of transforming comprises a method selected from the group consisting of microprojectile bombardment, PEG mediated transformation of protoplasts, electroporation, silicon carbide fiber mediated transformation, or Agrobacterium-mediated transformation. In another embodiment the recipient plant cell is from a monocotyledonous plant, including a wheat, maize, rye, rice, turfgrass, oat, barley, sorghum, millet, or sugarcane plant. In further embodiments, the recipient plant cell is from a dicotyledonous plant, including a tobacco, tomato, potato, soybean, canola, sunflower, alfalfa or cotton plant. The selected protein can be any protein, for example, an insect resistance protein, a bacterial disease resistance protein, a fungal disease resistance protein, a viral disease resistance protein, a nematode disease resistance protein, a herbicide resistance protein, a protein affecting grain composition or quality, a nutrient utilization gene, a mycotoxin reduction protein, a male sterility protein, a selectable marker protein, a screenable marker protein, a negative selectable marker gene, a gene affecting plant agronomic characteristics, or an environment or stress resistance protein. The construct also may comprise an enhancer, for example, a rice actin 1 intron or rice actin 2 intron. The selected coding region also may be operably linked to a terminator, for example, a rice or other rbcS terminator.
In still yet another aspect, the invention provides a method of plant breeding comprising the steps of: (i) obtaining a transgenic plant comprising a selected DNA comprising a maize RS81 promoter; and (ii) crossing said transgenic plant with itself or a second plant. In one embodiment of the invention, the transgenic plant is a monocotyledonous plant, for example, a wheat, maize, oat, barley, rye, rice, turfgrass, sorghum, millet or sugarcane plant. In another embodiment of the invention, the transgenic plant is a dicotyledonous plant, for example, a tobacco, tomato, potato, soybean, canola, sunflower, alfalfa or cotton plant. In still another embodiment of the invention, the maize RS81 promoter is isolatable from the nucleic acid sequence of SEQ ID NO:1. The maize RS81 promoter may also comprise from about 100 to about 2584, from about 150 to about 2584, from about 250 to about 2584, from about 400 to about 2584, from about 600 to about 2584, from about 800 to about 2584, from about 1000 to about 2584, from about 1500 to about 2584, or from about 2000 to about 2584 contiguous nucleotides of the nucleic acid sequence of SEQ ID NO:1. The maize RS81 promoter may also comprise the nucleic acid sequence of SEQ ID NO:1.
In the method of plant breeding, the transgenic plant may be crossed with said second plant, and said second plant may be an inbred plant. The method may also further comprise the steps of: (iii) collecting seeds resulting from said crossing; (iv) growing said seeds to produce progeny plants; (v) identifying a progeny plant comprising said selected DNA; and (vi) crossing said progeny plant with itself or a third plant. In the method, said progeny plant may inherit the selected DNA through a male or female parent. In one embodiment of the invention, the second plant and said third plant are of the same genotype, and may be inbred plants.
The selected DNA may further comprise potentially any protein, including an insect resistance protein, a bacterial disease resistance protein, a fungal disease resistance protein, a viral disease resistance protein, a nematode disease resistance protein, a herbicide resistance protein, a protein affecting grain composition or quality, a nutrient utilization protein, a mycotoxin reduction protein, an environment or stress resistance protein, a male sterility protein, a selectable marker protein, a screenable marker protein, a negative selectable marker protein, or a protein affecting plant agronomic characteristics. The selected DNA may further comprise a genetic element which enhances the expression of said protein in said transgenic plant, for example, a rice actin 1 intron or rice actin 2 intron.